1. Field of the Invention
The invention relates generally to prostate specific antigen (PSA) and specifically to new forms of PSA that are specific for benign prostatic hyperplasia and methods of using the new forms of PSA.
2. Description of the Prior Art
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Prostate cancer is the most frequently diagnosed cancer in American males. Prostate specific antigen (PSA) has been widely used as a reliable prognostic marker in the management of patients with prostate cancer (1-3). PSA is a member of the human kallikrein family of serine proteases (extensively reviewed in (4)). It is a serine endopeptidase with chymotrypsin-like enzymatic activity. The mature form of PSA (identified as SEQ ID NO:1) has isoleucine as the N-terminal and 237 amino acid residues with a molecular mass of 28,400 D (5; 6).
PSA exists in the serum as the free form of PSA but the majority of the PSA is in a complex with xcex11-antichymotrypsin (ACT). It is generally accepted that the free PSA in serum is enzymatically inactive. PSA is a serine protease which is capable of complex formation with serum protease inhibitors. Human serum contains high levels of ACT and a2macroglobulin, both of which have been shown to complex with PSA (8). 70-95% of the PSA in serum, which can be detected by immunoassay, is in a complex with ACT. The remainder is non-complexed, free PSA (9; 10).
More recently it has been demonstrated that the level of free or non-complexed PSA in serum can improve the discrimination of prostate cancer from BPH (9-11). An elevated ratio of free PSA to total PSA (free plus complexed PSA) is more highly correlated with BPH. The reasons for the presence of free PSA in serum has therefore become the subject of intensive investigation.
Studies with PSA purified from seminal plasma have shown that about 30% of the PSA does not form a complex with ACT. This fraction of PSA contains an internal peptide bond cleavage at Lysine 145 which renders it inactive (8-10). A more detailed analysis of the inactive forms of PSA from seminal plasma revealed PSA clipped at both Lysine 145 and Arginine 85, in addition to a fraction of PSA which was not clipped but which did not form a complex with ACT (12).
One of the earliest investigations for purifying and characterizing PSA from seminal plasma reported the presence of internal clips at residues Arginine 85, Lysine 148 and Lysine 185 (5). However, the relative proportion and significance of these clips was not determined at that time. Subsequent studies of inactive PSA, as cited above, have focused almost exclusively on the clip at Lysine 145, since this is the predominant clip in seminal plasma PSA and does in fact render PSA inactive. There is no suggestion in these studies that the Lysine 145 clip represents anything more than a random physiological cleavage which occurs some time after PSA expression. It is suggested that this clip could account for the presence of free PSA in serum. There is, however, no evidence that PSA in seminal plasma is representative of PSA found in serum, since serum PSA is due to the retrograde release of PSA from prostate tissues (4).
PSA has also been isolated from BPH tissue in the prostate in order to determine whether this form of PSA was different from seminal plasma PSA (13). PSA from BPH tissue was found to be less enzymatically active than seminal plasma PSA and to contain additional clips, including clips after Ile1, His54, Phe57 and Lys146. The characterization of PSA from BPH tissue had three major conclusions: 1) the BPH PSA is more clipped than seminal plasma PSA; 2) the BPH PSA is less enzymatically active than seminal plasma PSA; and 3) BPH PSA contains some unusual clips not found in seminal plasma PSA.
The limitation of this approach is that BPH tissue PSA was not compared to other matched prostate tissues but rather to seminal plasma PSA. It was not established whether BPH PSA was less enzymatically active than, or different from, other prostate tissue PSA. This is an important experimental consideration, since the preparation of PSA from tissues could include proteases from tissues not found in seminal plasma, or vice versa. In theory, PSA prepared from any prostate tissue might be more inactive than seminal plasma PSA, due merely to the presence of selective endogenous proteolysis, or proteolysis during tissue homogenization and PSA purification.
The major limitation of a PSA test is its lack of specificity to distinguish between benign prostatic hyperplasia (BPH) and prostate cancer (7). Elevated levels of PSA in the blood are symptomatic of prostate disease, which is primarily manifested as either BPH or prostate cancer. However, levels in the range of 4-10 ng PSA per ml of serum make BPH difficult to distinguish from prostate cancer without additional tests, such as a digital rectal exam and prostate needle biopsy. Clearly, a need exists to develop a serum marker that is specific for BPH. A need also exists to develop a diagnostic method that may be used to distinguish BPH from prostate cancer.
The present invention takes a different approach than previous approaches by other investigators in that the present invention has focused exclusively on prostate tissues as the source of variable forms of PSA in serum. In order to determine what molecular forms of PSA are present in the prostate, the present invention examined three different types of prostate tissue: 1) non-cancerous peripheral zone (PZ-N) tissue; 2) cancerous PZ tissue containing at least 80% tumor (PZ-C); and 3) non-cancerous transitional zone (TZ) tissue. It is the TZ which becomes hyperplastic in patients with BPH. In contrast, most cancers are found in the PZ. The present invention discovers that different forms of PSA are present in different types of tissues.
The present invention is based on the unexpected discovery of novel forms of PSA existing at an elevated level in human prostate transition zone (TZ) which contains the benign prostatic hyperplasia tissue. One novel form of PSA in the present invention includes at least one clip at Lysine 182 of the amino acid sequence of a mature form of PSA identified by SEQ ID NO:1. A novel form of PSA of the present invention may also include an additional one or more clips at Ile 1, Lys 145 and Lys 146 of the amino acid sequence of a mature form of PSA identified by SEQ ID NO:1. The novel forms of PSA have a unique conformation of their own and can be separated from a mature form of PSA or other forms of PSA by high performance hydrophobic interaction chromatography (HIC-HPLC). It is the discovery of the present invention that the novel forms of PSA exist at an elevated level in BPH tissues. Therefore, the novel forms of PSA of the present invention may be used as a serum marker or an immunohistological marker to distinguish BPH tissues.
Accordingly, one aspect of the present invention provides a substantially pure and isolated form of prostate specific antigen (PSA) comprising at least one clip at Lysine 182 of the amino acid sequence of a mature form of PSA identified by SEQ ID NO:1. The form of PSA may also include an additional one or more clips at Ile 1, Lys 145 and Lys 146 of the amino acid sequence of a mature PSA. In one embodiment, the novel form of PSA of the present invention consists of two clips at Lys 14 and Lys 182 of amino acid sequence of a mature form of PSA.
Another aspect of the present invention provides an antibody that is preferentially reactive with novel forms of PSA of the present invention. The antibody may be a monoclonal or polyclonal antibody. Immunogens, cell lines and methods of making the preferential reactive antibodies are also provided.
A further aspect of the present invention provides a method of detecting or determining in a sample a novel form of PSA of the present invention. The method includes the steps of:
(a) contacting an amount of an agent which specifically binds to the form of PSA to be detected with the sample under a condition that allows the formation of a binary complex comprising the agent and the form of PSA; and
(b) detecting or determining the presence or amount of the complex.
In accordance with embodiments of the present invention, the sample may be a human physiological fluid or a tissue specimen. The agent may be an antibody, and the antibody may be labeled or able to bind to a label, or the antibody may be bound to a solid phase.
Yet another aspect of the present invention provides a diagnostic kit for detecting or determining in a sample a novel form of PSA of the present invention. Also provided is a diagnostic method for distinguishing BPH tissues. A competitive immunoassay is also provided for detecting a novel form of PSA of the present invention.
The invention is defined in its fullest scope in the appended claims and is described below in its preferred embodiments.